Acquisition and maintenance of motility are important components of sperm physiology in that they play critical roles in ensuring fertility. While the roles that structural/motor proteins and ion channels play in sperm motility is under intense investigation and is beginning to become clear, the roles that active membrane transporters play in regulating sperm motility is not as well understood. However, recent evidence suggests that the Na/H exchanger (NHE) is important in maintaining an optimal intracellular pH critical for sperm motility. In agreement with this hypothesis, knockout of the sperm-specific NHE, NHE10, results in immotile mouse sperm confirming a link between NHE activity and sperm motility. The motility of these NHE10 knockout sperm can be restored either by increasing intracellular pH or by increasing intracellular cAMP concentration. Our preliminary studies demonstrate that in order for cAMP to restore motility to acidified sperm, an active NHE is required. Towards defining which of the three sperm NHE isoforms is required for cAMP to restore motility, we made the exciting discovery that an inhibitor of the NHE, by itself, can completely restore motility to NHE10 knockout sperm. In the studies proposed here, we will define the intracellular signaling molecules participating in the Camp dependent, motility-restoring pathway activated by the NHE inhibitor, we will identify which of the three NHE found in sperm initiate this pathway, and which of these isoforms serves as the ultimate downstream effectors to restore motility. These studies will not only be important for defining the role that the NHEs play in regulating sperm motility but they also may have medically relevant implications: Human sperm also contains this NHE inhibitor initiated motility-regulating signaling pathway and therefore a subset of infertile men may be infertile due to disruption in this pathway and specific inhibitors or activators of this pathway could be developed as therapies for male fertility/infertility treatments. PUBLIC HEALTH RELEVANCE: It is well accepted that adequate sperm motility is one of the key components of male fertility. Individuals with poorly motile or immotile sperm are typically infertile. In the US alone an estimated 10-15% of couples are considered infertile as defined by World Health Organization criteria. Among them, it is estimated that 30% of these couples are infertile due to the male partner. Therefore, a thorough understanding of the molecular events that allows sperm to acquire and maintain motility will help us to address issues related to infertility associated with poorly motile or immotile sperm. In addition, the knowledge gained in understanding the molecules involved in maintaining sperm motility could be useful in developing targets for effective male contraception.